Introduction The efficacy of prolonged-release fampridine (PR-FAM) may extend in multiple sclerosis (MS) beyond walking ability. The objective of this study was to evaluate the effect of PR-FAM treatment on cognition, fatigue, depression, and quality of life (QoL) in adult patients with MS in a real-world setting. Methods FAMILY was a multi-center, prospective, observational, real-world cohort study of MS patients receiving PR-FAM in the outpatient setting. Patients were treated as per PR-FAM’s local prescribing information for 6 months. Standardized protocols and questionnaires were used to evaluate changes in cognition (PASAT; Paced Auditory Serial Addition Test), fatigue (MFIS; Modified Fatigue Impact Scale), depression (BDI-II; Beck Depression Inventory-II) and QoL (MusiQoL; MS International Quality-of-Life questionnaire, MSIS-29; Multiple Sclerosis Impact Scale: PHYS and PSYCH subscales) at 3 and 6 months compared to baseline. Results In total, 102 eligible patients from 8 sites in Greece were analysed, of whom 92 completed the study and 10 discontinued. At 6 months, PR-FAM treatment resulted in improvements from baseline in PASAT-3′′ ( p = 0.044), MFIS ( p < 0.001), BDI-II ( p < 0.001), MusiQoL ( p < 0.001) and MSIS-29-PHYS ( p = 0.012) and MSIS-PSYCH ( p < 0.001). A positive effect was evident already at 3 months in PASAT-3′′ (ns), MFIS ( p = 0.020), BDI-II ( p = 0.034), MusiQoL ( p = 0.001), MSIS-29-PHYS (ns) and MSIS-29-PSYCH ( p < 0.001). Conclusions This observational study provides new data to the current literature in support of PR-FAM’s positive effects in cognition, fatigue, depression, and QoL in a large, heterogeneous group of Greek MS patients in the real-world setting. Trial Registration ClinicalTrials.gov identifier, NCT03164018. Supplementary Material The online version of this article (10.1007/s12325-020-01606-5) contains supplementary material, which is available to authorized users.